Pyrazole derivatives as sphingosine 1-phosphate (s1p) receptor modulators

ABSTRACT

The present invention relates to pyrazole derivatives, processes for preparing them, pharmaceutical compositions containing them and their use as pharmaceuticals as modulators of sphingosine-1-phosphate receptors.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 61/768,948 filed Feb. 25, 2013, the disclosure ofwhich is hereby incorporated in its entirety herein by reference.

FIELD OF THE INVENTION

The present invention relates to pyrazole derivatives, processes forpreparing them, pharmaceutical compositions containing them and theiruse as pharmaceuticals as modulators of sphingosine-1-phosphatereceptors. The invention relates specifically to the use of thesecompounds and their pharmaceutical compositions to treat disordersassociated with sphingosine-1-phosphate (S1P) receptor modulation.

BACKGROUND OF THE INVENTION

Sphingosine-1 phosphate is stored in relatively high concentrations inhuman platelets, which lack the enzymes responsible for its catabolism,and it is released into the blood stream upon activation ofphysiological stimuli, such as growth factors, cytokines, and receptoragonists and antigens. It may also have a critical role in plateletaggregation and thrombosis and could aggravate cardiovascular diseases.On the other hand the relatively high concentration of the metabolite inhigh-density lipoproteins (HDL) may have beneficial implications foratherogenesis. For example, there are recent suggestions thatsphingosine-1-phosphate, together with other lysolipids such assphingosylphosphorylcholine and lysosulfatide, are responsible for thebeneficial clinical effects of HDL by stimulating the production of thepotent antiatherogenic signaling molecule nitric oxide by the vascularendothelium. In addition, like lysophosphatidic acid, it is a marker forcertain types of cancer, and there is evidence that its role in celldivision or proliferation may have an influence on the development ofcancers. These are currently topics that are attracting great interestamongst medical researchers, and the potential for therapeuticintervention in sphingosine-1-phosphate metabolism is under activeinvestigation.

SUMMARY OF THE INVENTION

A group of novel pyrazole derivatives which are potent and selectivesphingosine-1-phosphate modulators has been discovered. As such, thecompounds described herein are useful in treating a wide variety ofdisorders associated with modulation of sphingosine-1-phosphatereceptors. The term “modulator” as used herein, includes but is notlimited to: receptor agonist, antagonist, inverse agonist, inverseantagonist, partial agonist, partial antagonist.

This invention describes compounds of Formula I, which havesphingosine-1-phosphate receptor biological activity. The compounds inaccordance with the present invention are thus of use in medicine, forexample in the treatment of humans with diseases and conditions that arealleviated by S1P modulation.

In one aspect, the invention provides a compound having Formula I or apharmaceutically acceptable salt thereof or stereoisomeric formsthereof, or the geometrical isomers, enantiomers, diastereoisomers,tautomers, zwitterions and pharmaceutically acceptable salts thereof:

wherein:R is H, optionally substituted C₁₋₆ alkyl, —OC₁₋₃ alkyl, halogen,optionally substituted C₆₋₁₀ aryl, optionally substituted heterocycle,CN, NO₂, C(O)R⁵, NR⁶R⁷ or OH;R¹ is H, halogen, optionally substituted C₁₋₆ alkyl, halogen, optionallysubstituted C₆₋₁₀ aryl, optionally substituted heterocycle;R² is H, halogen, optionally substituted C₁₋₆ alkyl, halogen, optionallysubstituted C₆₋₁₀ aryl, optionally substituted heterocycle;R³ is H, optionally substituted C₁₋₆ alkyl, NR⁶R⁷ or hydroxyl;R⁴ is H, optionally substituted C₁₋₆ alkyl;R⁵ is H, OH or optionally substituted C₁₋₆ alkyl;a is 0, 1, 2 or 3;b is 0, 1, 2 or 3;

X is S, O, NR⁶ or CHR⁷;

R⁶ is H, optionally substituted C₁₋₆ alkyl;R⁷ is H, optionally substituted C₁₋₆ alkyl;Y is OPO₃H₂, carboxylic acid, PO₃H₂, —P(O)MeOH, —P(O)(H)OH, NR⁸R⁹ orOR¹⁰;

R⁸ is H, —C(O)CH₂OH or —CH₂COOH; R⁹ is H, —C(O)CH₂OH or —CH₂COOH; and

R¹⁰ is H or optionally substituted C₁₋₃ alkyl.

In another aspect the invention provides a compound of Formula I,wherein:

R is H, optionally substituted C₁₋₆ alkyl or halogen;R¹ is H, halogen or optionally substituted C₁₋₆ alkyl;R² is H, halogen or optionally substituted C₁₋₆ alkyl;R³ is H, NR⁶R⁷ or hydroxyl;R⁴ is H or optionally substituted C₁₋₆ alkyl;a is 0 or 1;b is 0 or 1;

X is O, NR⁶ or CHR⁷; R⁶ is H; R⁷ is H;

Y is OPO₃H₂, carboxylic acid, PO₃H₂, NR⁸R⁹ or OR¹⁰;

R⁸ is H, —C(O)CH₂OH or —CH₂COOH; R⁹ is H, —C(O)CH₂OH or —CH₂COOH; andR¹⁰ is H.

In another aspect the invention provides a compound of Formula I,wherein:

R is H;

R¹ is H or optionally substituted C₁₋₆ alkyl;R² is H or optionally substituted C₁₋₆ alkyl;R³ is H or hydroxyl;R⁴ is H or methyl;a is 0 or 1;b is 0 or 1;

X is O or NR⁶; R⁶ is H;

Y is OPO₃H₂, carboxylic acid, PO₃H₂, NR⁸R⁹ or OR¹⁰;

R⁸ is H, —C(O)CH₂OH or —CH₂COOH; R⁹ is H; and R¹⁰ is H.

In another aspect the invention provides a compound of Formula I,wherein:

R is H;

R¹ is H or methyl;R² is H or methyl;R³ is H or hydroxyl;R⁴ is methyl;a is 0 or 1;b is 0 or 1;

X is O or NR⁶; R⁶ is H;

Y is OPO₃H₂, carboxylic acid, PO₃H₂, NR⁸R⁹ or OR¹⁰;

R⁸ is H, —C(O)CH₂OH or —CH₂COOH; R⁹ is H; and R¹⁰ is H.

In another aspect the invention provides a compound of Formula I,wherein:

R is H;

R¹ is H or methyl;R² is H or methyl;R³ is H or hydroxyl;R⁴ is methyl;a is 1;b is 0;

X is NR⁶; R⁶ is H; and

Y is carboxylic acid.

In another aspect the invention provides a compound of Formula I,wherein:

R is H;

R¹ is H or methyl;R² is H or methyl;R³ is H or hydroxyl;R⁴ is methyl;a is 1;b is 0 or 1;

X is NR⁶; R⁶ is H;

Y is OPO₃H₂, carboxylic acid, PO₃H₂, NR⁸R⁹ or OR¹⁰;

R⁸ is H, —C(O)CH₂OH or —CH₂COOH; R⁹ is H; and R¹⁰ is H.

In another aspect the invention provides a compound of Formula I,wherein:

R is H;

R¹ is H or methyl;R² is H or methyl;R³ is H or hydroxyl;R⁴ is methyl;a is 0;b is 0 or 1;

X is O;

Y is OPO₃H₂, carboxylic acid, PO₃H₂, NR⁸R⁹ or OR¹⁰;

R⁸ is H, —C(O)CH₂OH or —CH₂COOH; R⁹ is H; and R¹⁰ is H.

In another aspect the invention provides a compound of Formula I,wherein:

R is H;

R¹ is methyl;R² is methyl;R³ is H or hydroxyl;R⁴ is methyl;a is 0;b is 0 or 1;

X is O; Y is OPO₃H₂, NR⁸R⁹ or OR¹⁰; R⁸ is H, —C(O)CH₂OH or —CH₂COOH; R⁹is H; and R¹⁰ is H.

In another aspect the invention provides a compound of Formula I,wherein:

R is H, R¹ is H; R² is H; R³ is H;

R⁴ is methyl;a is 1;b is 0 or 1;

X is NR⁶; R⁶ is H; and

Y is carboxylic acid or PO₃H₂.

The term “alkyl”, as used herein, refers to saturated, monovalent ordivalent hydrocarbon moieties having linear or branched moieties orcombinations thereof and containing 1 to 6 carbon atoms. One methylene(—CH₂—) group, of the alkyl can be replaced by oxygen, sulfur,sulfoxide, nitrogen, carbonyl, carboxyl, sulfonyl, or by a divalent C₃₋₆cycloalkyl. Alkyl groups can be substituted by halogen, hydroxyl,cycloalkyl, amino, non-aromatic heterocycles, carboxylic acid,phosphonic acid groups, sulphonic acid groups, phosphoric acid.

The term “cycloalkyl”, as used herein, refers to a monovalent ordivalent group of 3 to 8 carbon atoms, derived from a saturated cyclichydrocarbon. Cycloalkyl groups can be monocyclic or polycyclic.Cycloalkyl can be substituted by C₁₋₃ alkyl groups or halogens.

The term “halogen”, as used herein, refers to an atom of chlorine,bromine, fluorine, iodine.

The term “heterocycle” as used herein, refers to a 3 to 10 memberedring, which can be aromatic or non-aromatic, saturated or non-saturated,containing at least one heteroatom selected form O or N or S orcombinations of at least two thereof, interrupting the carbocyclic ringstructure. Heterocycle can be monocyclic or polycyclic. The heterocyclicring can be interrupted by a C═O; the S heteroatom can be oxidized.Heterocycles can be monocyclic or polycyclic. Heterocyclic ring moietiescan be substituted by hydroxyl, C₁₋₃ alkyl or halogens.

The term “aryl” as used herein, refers to an organic moiety derived froman aromatic hydrocarbon consisting of a ring containing 6 to 10 carbonatoms by removal of one hydrogen. Aryl can be monocyclic or polycyclic.Aryl can be substituted by halogen atoms, —OC₁₋₃ alkyl, C₁₋₃ alkyl, CN,—C(O)H or —C(O)(C₁₋₃ alkyl), NH(C₁₋₃ alkyl), NH₂, N(C₁₋₃ alkyl) (C₁₋₃alkyl), NO₂ or hydroxyl. Usually aryl is phenyl. Preferred substitutionsite on aryl are meta and para positions.

The term “hydroxyl” as used herein, represents a group of formula “—OH”.

The term “carbonyl” as used herein, represents a group of formula“—C(O)”.

The term “carboxyl” as used herein, represents a group of formula“—C(O)O—”.

The term “sulfonyl” as used herein, represents a group of formula“—SO₂”.

The term “sulfate” as used herein, represents a group of formula“—O—S(O)₂—O—”.

The term “carboxylic acid” as used herein, represents a group of formula“—C(O)ON”.

The term “sulfoxide” as used herein, represents a group of formula“—S═O”.

The term “phosphonic acid” as used herein, represents a group of formula“—P(O)(OH)₂”.

The term “phosphoric acid” as used herein, represents a group of formula“—(O)P(O)(OH)₂”.

The term “sulphonic acid” as used herein, represents a group of formula“—S(O)₂OH”.

The formula “H”, as used herein, represents a hydrogen atom.

The formula “O”, as used herein, represents an oxygen atom.

The formula “N”, as used herein, represents a nitrogen atom.

The formula “S”, as used herein, represents a sulfur atom.

Compounds of the invention are:

Some compounds of Formula I and some of their intermediates have atleast one stereogenic center in their structure. This stereogenic centermay be present in an R or S configuration, said R and S notation is usedin correspondence with the rules described in Pure Appli. Chem. (1976),45, 11-13.

The term “pharmaceutically acceptable salts” refers to salts orcomplexes that retain the desired biological activity of the aboveidentified compounds and exhibit minimal or no undesired toxicologicaleffects. The “pharmaceutically acceptable salts” according to theinvention include therapeutically active, non-toxic base or acid saltforms, which the compounds of Formula I are able to form.

The acid addition salt form of a compound of Formula I that occurs inits free form as a base can be obtained by treating the free base withan appropriate acid such as an inorganic acid, for example, hydrochloricacid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid andthe like; or an organic acid such as for example, acetic, hydroxyacetic,propanoic, lactic, pyruvic, malonic, fumaric acid, maleic acid, oxalicacid, tartaric acid, succinic acid, malic acid, ascorbic acid, benzoicacid, tannic acid, pamoic acid, citric, methylsulfonic, ethanesulfonic,benzenesulfonic, formic and the like (Handbook of Pharmaceutical Salts,P. Heinrich Stahl & Camille G. Wermuth (Eds), Verlag Helvetica ChimicaActa—Zürich, 2002, 329-345).

Compounds of Formula I and their salts can be in the form of a solvate,which is included within the scope of the present invention. Suchsolvates include for example hydrates, alcoholates and the like.

With respect to the present invention reference to a compound orcompounds, is intended to encompass that compound in each of itspossible isomeric forms and mixtures thereof unless the particularisomeric form is referred to specifically.

Compounds according to the present invention may exist in differentpolymorphic forms. Although not explicitly indicated in the aboveformula, such forms are intended to be included within the scope of thepresent invention.

The compounds of the invention are indicated for use in treating orpreventing conditions in which there is likely to be a componentinvolving the sphingosine-1-phosphate receptors.

In another embodiment, there are provided pharmaceutical compositionsincluding at least one compound of the invention in a pharmaceuticallyacceptable carrier.

In a further embodiment of the invention, there are provided methods fortreating disorders associated with modulation of sphingosine-1-phosphatereceptors. Such methods can be performed, for example, by administeringto a subject in need thereof a pharmaceutical composition containing atherapeutically effective amount of at least one compound of theinvention.

These compounds are useful for the treatment of mammals, includinghumans, with a range of conditions and diseases that are alleviated byS1P modulation: not limited to the treatment of diabetic retinopathy,other retinal degenerative conditions, dry eye, angiogenesis and wounds.

-   -   Therapeutic utilities of S1P modulators are Ocular Diseases: wet        and dry age-related macular degeneration, diabetic retinopathy,        retinopathy of prematurity, retinal edema, geographic atrophy,        glaucomatous optic neuropathy, chorioretinopathy, hypertensive        retinopathy, ocular ischemic syndrome, prevention of        inflammation-induced fibrosis in the back of the eye, various        ocular inflammatory diseases including uveitis, scleritis,        keratitis, and retinal vasculitis;    -   Systemic vascular barrier related diseases: various inflammatory        diseases, including acute lung injury, its prevention, sepsis,        tumor metastasis, atherosclerosis, pulmonary edemas, and        ventilation-induced lung injury;    -   Autoimmune diseases and immunosuppression: rheumatoid arthritis,        Crohn's disease, Graves' disease, inflammatory bowel disease,        multiple sclerosis, Myasthenia gravis, Psoriasis, ulcerative        colitis, autoimmune uveitis, renal ischemia/perfusion injury,        contact hypersensitivity, atopic dermatitis, and organ        transplantation;    -   Allergies and other inflammatory diseases: urticaria, bronchial        asthma, and other airway inflammations including pulmonary        emphysema and chronic obstructive pulmonary diseases;    -   Cardiac functions: bradycardia, congestional heart failure,        cardiac arrhythmia, prevention and treatment of atherosclerosis,        and ischemia/reperfusion injury;    -   Wound Healing: scar-free healing of wounds from cosmetic skin        surgery, ocular surgery, GI surgery, general surgery, oral        injuries, various mechanical, heat and burn injuries, prevention        and treatment of photoaging and skin ageing, and prevention of        radiation-induced injuries;    -   Bone formation: treatment of osteoporosis and various bone        fractures including hip and ankles;    -   Anti-nociceptive activity: visceral pain, pain associated with        diabetic neuropathy, rheumatoid arthritis, chronic knee and        joint pain, tendonitis, osteoarthritis, neuropathic pains;    -   Anti-fibrosis: ocular, cardiac, hepatic and pulmonary fibrosis,        proliferative vitreoretinopathy, cicatricial pemphigoid,        surgically induced fibrosis in cornea, conjunctiva and tenon;    -   Pains and anti-inflammation: acute pain, flare-up of chronic        pain, musculo-skeletal pains, visceral pain, pain associated        with diabetic neuropathy, rheumatoid arthritis, chronic knee and        joint pain, tendonitis, osteoarthritis, bursitis, neuropathic        pains;    -   CNS neuronal injuries: Alzheimer's disease, age-related neuronal        injuries;    -   Organ transplants: renal, corneal, cardiac and adipose tissue        transplants.

In still another embodiment of the invention, there are provided methodsfor treating disorders associated with modulation ofsphingosine-1-phosphate receptors. Such methods can be performed, forexample, by administering to a subject in need thereof a therapeuticallyeffective amount of at least one compound of the invention, or anycombination thereof, or pharmaceutically acceptable salts, hydrates,solvates, crystal forms and individual isomers, enantiomers, anddiastereisomers thereof.

-   -   The present invention concerns the use of a compound of Formula        I or a pharmaceutically acceptable salt thereof, for the        manufacture of a medicament for the treatment of Ocular        Diseases: wet and dry age-related macular degeneration, diabetic        retinopathy, retinopathy of prematurity, retinal edema,        geographic atrophy, glaucomatous optic neuropathy,        chorioretinopathy, hypertensive retinopathy, ocular ischemic        syndrome, prevention of inflammation-induced fibrosis in the        back of the eye, various ocular inflammatory diseases including        uveitis, scleritis, keratitis, and retinal vasculitis;    -   Systemic vascular barrier related diseases: various inflammatory        diseases, including acute lung injury, its prevention, sepsis,        tumor metastasis, atherosclerosis, pulmonary edemas, and        ventilation-induced lung injury;    -   Autoimmune diseases and immunosuppression: rheumatoid arthritis,        Crohn's disease, Graves' disease, inflammatory bowel disease,        multiple sclerosis, Myasthenia gravis, Psoriasis, ulcerative        colitis, autoimmune uveitis, renal ischemia/perfusion injury,        contact hypersensitivity, atopic dermatitis, and organ        transplantation;    -   Allergies and other inflammatory diseases: urticaria, bronchial        asthma, and other airway inflammations including pulmonary        emphysema and chronic obstructive pulmonary diseases;    -   Cardiac functions: bradycardia, congestional heart failure,        cardiac arrhythmia, prevention and treatment of atherosclerosis,        and ischemia/reperfusion injury;    -   Wound Healing: scar-free healing of wounds from cosmetic skin        surgery, ocular surgery, GI surgery, general surgery, oral        injuries, various mechanical, heat and burn injuries, prevention        and treatment of photoaging and skin ageing, and prevention of        radiation-induced injuries;    -   Bone formation: treatment of osteoporosis and various bone        fractures including hip and ankles;    -   Anti-nociceptive activity: visceral pain, pain associated with        diabetic neuropathy, rheumatoid arthritis, chronic knee and        joint pain, tendonitis, osteoarthritis, neuropathic pains;    -   Anti-fibrosis: ocular, cardiac, hepatic and pulmonary fibrosis,        proliferative vitreoretinopathy, cicatricial pemphigoid,        surgically induced fibrosis in cornea, conjunctiva and tenon;    -   Pains and anti-inflammation: acute pain, flare-up of chronic        pain, musculo-skeletal pains, visceral pain, pain associated        with diabetic neuropathy, rheumatoid arthritis, chronic knee and        joint pain, tendonitis, osteoarthritis, bursitis, neuropathic        pains;    -   CNS neuronal injuries: Alzheimer's disease, age-related neuronal        injuries;    -   Organ transplants: renal, corneal, cardiac and adipose tissue        transplants.

The actual amount of the compound to be administered in any given casewill be determined by a physician taking into account the relevantcircumstances, such as the severity of the condition, the age and weightof the patient, the patient's general physical condition, the cause ofthe condition, and the route of administration.

The patient will be administered the compound orally in any acceptableform, such as a tablet, liquid, capsule, powder and the like, or otherroutes may be desirable or necessary, particularly if the patientsuffers from nausea. Such other routes may include, without exception,transdermal, parenteral, subcutaneous, intranasal, via an implant stent,intrathecal, intravitreal, topical to the eye, back to the eye,intramuscular, intravenous, and intrarectal modes of delivery.Additionally, the formulations may be designed to delay release of theactive compound over a given period of time, or to carefully control theamount of drug released at a given time during the course of therapy.

In another embodiment of the invention, there are providedpharmaceutical compositions including at least one compound of theinvention in a pharmaceutically acceptable carrier thereof. The phrase“pharmaceutically acceptable” means the carrier, diluent or excipientmust be compatible with the other ingredients of the formulation and notdeleterious to the recipient thereof.

Pharmaceutical compositions of the present invention can be used in theform of a solid, a solution, an emulsion, a dispersion, a patch, amicelle, a liposome, and the like, wherein the resulting compositioncontains one or more compounds of the present invention, as an activeingredient, in admixture with an organic or inorganic carrier orexcipient suitable for enteral or parenteral applications. Inventioncompounds may be combined, for example, with the usual non-toxic,pharmaceutically acceptable carriers for tablets, pellets, capsules,suppositories, solutions, emulsions, suspensions, and any other formsuitable for use. The carriers which can be used include glucose,lactose, gum acacia, gelatin, mannitol, starch paste, magnesiumtrisilicate, talc, corn starch, keratin, colloidal silica, potatostarch, urea, medium chain length triglycerides, dextrans, and othercarriers suitable for use in manufacturing preparations, in solid,semisolid, or liquid form. In addition auxiliary, stabilizing,thickening and coloring agents and perfumes may be used. Inventioncompounds are included in the pharmaceutical composition in an amountsufficient to produce the desired effect upon the process or diseasecondition.

Pharmaceutical compositions containing invention compounds may be in aform suitable for oral use, for example, as tablets, troches, lozenges,aqueous or oily suspensions, dispersible powders or granules, emulsions,hard or soft capsules, or syrups or elixirs. Compositions intended fororal use may be prepared according to any method known in the art forthe manufacture of pharmaceutical compositions and such compositions maycontain one or more agents selected from the group consisting of asweetening agent such as sucrose, lactose, or saccharin, flavoringagents such as peppermint, oil of wintergreen or cherry, coloring agentsand preserving agents in order to provide pharmaceutically elegant andpalatable preparations. Tablets containing invention compounds inadmixture with non-toxic pharmaceutically acceptable excipients may alsobe manufactured by known methods. The excipients used may be, forexample, (1) inert diluents such as calcium carbonate, lactose, calciumphosphate or sodium phosphate; (2) granulating and disintegrating agentssuch as corn starch, potato starch or alginic acid; (3) binding agentssuch as gum tragacanth, corn starch, gelatin or acacia, and (4)lubricating agents such as magnesium stearate, stearic acid or talc. Thetablets may be uncoated or they may be coated by known techniques todelay disintegration and absorption in the gastrointestinal tract andthereby provide a sustained action over a longer period. For example, atime delay material such as glyceryl monostearate or glyceryl distearatemay be employed.

In some cases, formulations for oral use may be in the form of hardgelatin capsules wherein the invention compounds are mixed with an inertsolid diluent, for example, calcium carbonate, calcium phosphate orkaolin. They may also be in the form of soft gelatin capsules whereinthe invention compounds are mixed with water or an oil medium, forexample, peanut oil, liquid paraffin or olive oil.

The pharmaceutical compositions may be in the form of a sterileinjectable suspension. This suspension may be formulated according toknown methods using suitable dispersing or wetting agents and suspendingagents. The sterile injectable preparation may also be a sterileinjectable solution or suspension in a non-toxic parenterally-acceptablediluent or solvent, for example, as a solution in 1,3-butanediol.Sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose any bland fixed oil may be employedincluding synthetic mono- or diglycerides, fatty acids (including oleicacid), naturally occurring vegetable oils like sesame oil, coconut oil,peanut oil, cottonseed oil, etc., or synthetic fatty vehicles like ethyloleate or the like. Buffers, preservatives, antioxidants, and the likecan be incorporated as required.

Pharmaceutical compositions containing invention compounds may be in aform suitable for topical use, for example, as oily suspensions, assolutions or suspensions in aqueous liquids or nonaqueous liquids, or asoil-in-water or water-in-oil liquid emulsions. Pharmaceuticalcompositions may be prepared by combining a therapeutically effectiveamount of at least one compound according to the present invention, or apharmaceutically acceptable salt thereof, as an active ingredient withconventional ophthalmically acceptable pharmaceutical excipients and bypreparation of unit dosage suitable for topical ocular use. Thetherapeutically efficient amount typically is between about 0.001 andabout 5% (w/v), preferably about 0.001 to about 2.0% (w/v) in liquidformulations.

For ophthalmic application, preferably solutions are prepared using aphysiological saline solution as a major vehicle. The pH of suchophthalmic solutions should preferably be maintained between 4.5 and 8.0with an appropriate buffer system, a neutral pH being preferred but notessential. The formulations may also contain conventionalpharmaceutically acceptable preservatives, stabilizers and surfactants.Preferred preservatives that may be used in the pharmaceuticalcompositions of the present invention include, but are not limited to,benzalkonium chloride, chlorobutanol, thimerosal, phenylmercuric acetateand phenylmercuric nitrate. A preferred surfactant is, for example,Tween 80. Likewise, various preferred vehicles may be used in theophthalmic preparations of the present invention. These vehiclesinclude, but are not limited to, polyvinyl alcohol, povidone,hydroxypropyl methyl cellulose, poloxamers, carboxymethyl cellulose,hydroxyethyl cellulose cyclodextrin and purified water.

Tonicity adjustors may be added as needed or convenient. They include,but are not limited to, salts, particularly sodium chloride, potassiumchloride, mannitol and glycerin, or any other suitable ophthalmicallyacceptable tonicity adjustor.

Various buffers and means for adjusting pH may be used so long as theresulting preparation is ophthalmically acceptable. Accordingly, buffersinclude acetate buffers, citrate buffers, phosphate buffers and boratebuffers. Acids or bases may be used to adjust the pH of theseformulations as needed.

In a similar manner an ophthalmically acceptable antioxidant for use inthe present invention includes, but is not limited to, sodiummetabisulfite, sodium thiosulfate, acetylcysteine, butylatedhydroxyanisole and butylated hydroxytoluene. Other excipient componentswhich may be included in the ophthalmic preparations are chelatingagents. The preferred chelating agent is edentate disodium, althoughother chelating agents may also be used in place of or in conjunctionwith it.

The ingredients are usually used in the following amounts:

Ingredient Amount (% w/v) active ingredient about 0.001 to about 5preservative   0-0.10 vehicle 0-40 tonicity adjustor 0-10 buffer0.01-10   pH adjustor q.s. pH 4.5-7.8 antioxidant as needed surfactantas needed purified water to make 100%

The actual dose of the active compounds of the present invention dependson the specific compound, and on the condition to be treated; theselection of the appropriate dose is well within the knowledge of theskilled artisan.

The ophthalmic formulations of the present invention are convenientlypackaged in forms suitable for metered application, such as incontainers equipped with a dropper, to facilitate application to theeye. Containers suitable for drop wise application are usually made ofsuitable inert, non-toxic plastic material, and generally containbetween about 0.5 and about 15 ml solution. One package may contain oneor more unit doses. Especially preservative-free solutions are oftenformulated in non-resalable containers containing up to about ten,preferably up to about five units doses, where a typical unit dose isfrom one to about 8 drops, preferably one to about 3 drops. The volumeof one drop usually is about 20-35 μl.

Invention compounds may also be administered in the form ofsuppositories for rectal administration of the drug. These compositionsmay be prepared by mixing the invention compounds with a suitablenon-irritating excipient, such as cocoa butter, synthetic glycerideesters of polyethylene glycols, which are solid at ordinarytemperatures, but liquefy and/or dissolve in the rectal cavity torelease the drug.

Since individual subjects may present a wide variation in severity ofsymptoms and each drug has its unique therapeutic characteristics, theprecise mode of administration and dosage employed for each subject isleft to the discretion of the practitioner.

The compounds and pharmaceutical compositions described herein areuseful as medicaments in mammals, including humans, for treatment ofdiseases and/or alleviations of conditions which are responsive totreatment by agonists or functional antagonists ofsphingosine-1-phosphate receptors. Thus, in further embodiments of theinvention, there are provided methods for treating a disorder associatedwith modulation of sphingosine-1-phosphate receptors. Such methods canbe performed, for example, by administering to a subject in need thereofa pharmaceutical composition containing a therapeutically effectiveamount of at least one invention compound. As used herein, the term“therapeutically effective amount” means the amount of thepharmaceutical composition that will elicit the biological or medicalresponse of a subject in need thereof that is being sought by theresearcher, veterinarian, medical doctor or other clinician. In someembodiments, the subject in need thereof is a mammal. In someembodiments, the mammal is human.

The present invention concerns also processes for preparing thecompounds of Formula I. The compounds of formula I according to theinvention can be prepared analogously to conventional methods asunderstood by the person skilled in the art of synthetic organicchemistry. The synthetic schemes set forth below, illustrate howcompounds according to the invention can be made.

The following abbreviations are used in Scheme 1 and in the examples:

NaIO₄ sodium periodateRuCl₃xH₂O ruthenium(II) chloride hydrate EtOAc ethylacetateNaCl sodium chlorideNaOH sodium hydroxideMgSO₄ magnesium sulfateDMF dimethylformideCsCO₃ cesium carbonateMeI methyl iodideNaOMe sodium methoxideMeOH methanolEt₂O diethyletherNaHCO₃ sodium bicarbonateCDCl₃ deuterated chloroformCH₂Cl₂ dichloromethaneMPLC medium pressure liquid chromatographyCD₃OD deuterated methanolNH₂NH₂—H₂O hydrazine monohydrateCDI 1,1′-carbonyldiimidazoleNaBH₃CN sodium cyanoborohydride

Those skilled in the art will be able to routinely modify and/or adaptthe following scheme to synthesize any compounds of the inventioncovered by Formula I.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention claimed. As used herein, theuse of the singular includes the plural unless specifically statedotherwise.

It will be readily apparent to those skilled in the art that some of thecompounds of the invention may contain one or more asymmetric centers,such that the compounds may exist in enantiomeric as well as indiastereisomeric forms. Unless it is specifically noted otherwise, thescope of the present invention includes all enantiomers, diastereomersand racemic mixtures. Some of the compounds of the invention may formsalts with pharmaceutically acceptable acids or bases, and suchpharmaceutically acceptable salts of the compounds described herein arealso within the scope of the invention.

The present invention includes all pharmaceutically acceptableisotopically enriched compounds. Any compound of the invention maycontain one or more isotopic atoms enriched or different than thenatural ratio such as deuterium ²H (or D) in place of hyrdrogen ¹H (orH) or use of ¹³C enriched material in place of ¹²O and the like. Similarsubstitutions can be employed for N, O and S. The use of isotopes mayassist in analytical as well as therapeutic aspects of the invention.For example, use of deuterium may increase the in vivo half-life byaltering the metabolism (rate) of the compounds of the invention. Thesecompounds can be prepared in accord with the preparations described byuse of isotopically enriched reagents.

The following examples are for illustrative purposes only and are notintended, nor should they be construed as limiting the invention in anymanner. Those skilled in the art will appreciate that variations andmodifications of the following examples can be made without exceedingthe spirit or scope of the invention.

As will be evident to those skilled in the art, individual isomericforms can be obtained by separation of mixtures thereof in conventionalmanner. For example, in the case of diasteroisomeric isomers,chromatographic separation may be employed.

Compound names were generated with ACDLabs version 12.5; and

Intermediates and reagent names used in the examples were generated withsoftware such as Chem Bio Draw Ultra version 12.0 or Auto Nom 2000 fromMDL ISIS Draw 2.5 SP1.

In general, characterization of the compounds is performed according tothe following methods. Proton nuclear magnetic resonance (¹H NMR) andcarbon nuclear magnetic resonance (¹³C NMR) spectra were recorded on aVarian 60 MHz spectrometer in deuterated solvent. Chemical shifts werereported as δ (delta) values in parts per million (ppm) relative totetramethylsilane (TMS) as an internal standard (0.00 ppm) andmultiplicities were reported as s, singlet; d, doublet; t, triplet; q,quartet; m, multiplet; br, broad. Data were reported in the followingformat: chemical shift (multiplicity, coupling constant(s) J in hertz(Hz), integrated intensity).

Example 1 Intermediate 12-(2,2-dimethyl-3-(2-oxopropyl)cyclopropyl)acetic acid

A 3-neck, 2 L flask equipped with a mechanical stirrer, condenser withArgon, thermometer, and temperature controlled heating mantle wascharged with t-butanol (157 mL), water (315 mL) and 3-carene (25 g,0.184 mol). To the stirred resultant cloudy suspension was added NaIO₄(140 g, 0.654 mol) and RuCl₃xH₂O (0.63 g, 0.003 mol). The vigorouslystirred thick mixture was heated at 35-45° C. for 2 hr, then at 45-50°C. for 1 hr and another 30 min at 55° C. The thick mixture was cooled to30° C. and filtered. The solid was pressed with dental dam and rinsedwith diethyl ether (200 mL). The original aqueous filtrate was extractedwith 2:1 EtOAc:Hexanes (250 mL). The organic layer and ether filtratewere combined and washed with 20% aqueous NaCl (85 mL), then extractedwith a solution of 10 g of NaOH in 620 mL of water. The aqueous layerwas cooled in an ice bath and acidified to pH 1 with 12 N HCl (28 mL)and extracted with diethyl ether (3×225 mL). The ether layers werecombined and washed with brine (110 mL). The organic layer was driedover anhydrous MgSO₄ (30 g) overnight. The organic layer was filteredand concentrated to give 20 g of Intermediate 1 as a brown oil (59%).

¹H NMR (60 MHz, CDCl₃): δ 11.4 (s, 1H), 2.2 (s, 3H), 2.5-2.1 (m, 4H),1.1 (s, 3H), 0.95 (s, 3H), 1.3-0.9 (m, 2H) ppm.

Example 2 Intermediate 2 Methyl2-(2,2-dimethyl-3-(2-oxopropyl)cyclopropyl)acetate

A 3-neck, 500 mL flask equipped with a stir bar, condenser, oil bath,and Ar inlet, was charged with Intermediate 1, (19.0 g, 0.10 mol), DMF(100 mL), and CsCO₃ (18.6 g, 0.057 mol). To the resultant mixture afterstirring 10 min was added MeI (6.86 mL, 0.11 mol). The mixture washeated at 40° C. for 1 hr. The reaction mixture was cooled with an icebath. The solids were filtered. The filtrate was quenched with 18% (w/w)aqueous NaCl (500 mL). The organic layer was separated and the aqueouslayer was extracted with 1:1 Et₂O:Hexanes (2×200 mL). The combinedorganic layers were washed with water (200 mL) and dried over anhydrousMgSO₄ (20 g) overnight. The organic layer was filtered and concentratedto give 15.6 g of Intermediate 2, as an orange oil (79%).

¹H NMR (60 MHz, CDCl₃): δ 3.6 (s, 3H), 2.4-2.3 (m, 4H), 2.2 (s, 3H), 1.1(s, 3H), 0.95 (s, 3H), 1.2-0.9 (m, 2H) ppm.

Example 3 Intermediate 3(Z)-2-(1-hydroxyethylidene)-6,6-dimethylbicyclo[3.1.0]hexan-3-one

A 3-neck, 250 mL flask equipped with a stir bar, condenser, oil bath,and Ar inlet, was charged with Intermediate 2, (15 g, 0.0757 mol) inmethanol (47 mL). To this solution was added 25% NaOMe in MeOH (28 mL,0.129 mol). The solution changed from orange to red. The reaction washeated 15 min and refluxed for 25 min. The mixture was cooled andextracted with toluene (2×25 mL). The aqueous layer was cooled in an icebath and 20% Aq HCl (25 mL) was added dropwise with stirring to pH 2.The aqueous layer was then extracted with Et₂O (2×100 mL). The etherlayers were combined and washed with 5% aqueous NaHCO₃ (2×25 mL)filtered through phase separation paper and concentrated to give 9.0 gof Intermediate 3, as a light orange oil (71%).

¹H NMR (60 MHz, CDCl₃): δ 2.6-2.3 (m, 2H), 2.0 (s, 3H), 1.9-1.6 (m, 1H),1.4-1.3 (m, 1H), 1.05 (s, 3H), 0.9 (s, 3H) ppm.

Example 4 Intermediate 43,4,4-trimethyl-3b,4,4a,5-tetrahydro-1H-cyclopropa[3,4]cyclopenta[1,2-c]pyrazole

A 3-neck, 250 mL flask equipped with a stir bar, condenser, and Arinlet, was charged with Intermediate 3, (9.0 g, 0.054 mol) in methanol(45 mL) and glacial acetic acid (9 mL) resulting in an orange solution.To this solution all at once was added hydrazine hydrate (3.15 g, 0.063mol). The solution was warm to the touch. After stirring for 2 hr atroom temperature no starting material was present by HPLC. The reactionwas cooled in an ice bath and 1M 50% NaOH was added dropwise to pH 9-10.A solid formed and was left stirring for 20 min, filtered, and dried togive 7.6 g of Intermediate 4, as an orange solid (87%).

¹H NMR (60 MHz, CDCl₃): δ 3.0-2.4 (m, 2H), 2.2 (s, 3H), 1.8-1.6 (m, 2H),1.0 (s, 3H), 0.6 (s, 3H) ppm.

Example 5 Intermediate 54-(3-oxo-3-(3,4,4-trimethyl-3b,4,4a,5-tetrahydro-1H-cyclopropa[3,4]cyclopenta[1,2-c]pyrazol-1-yl)propyl)benzaldehyde

A 3-neck, 250 mL flask equipped with a stir-bar, and Ar inlet, wascharged with 3-(4-formylphenyl)propionic acid (1.64 g, 0.0092 mol) inCH₂Cl₂ (40 mL). To this suspension was added all at once carbonyldiimidazole (1.57 g, 0.0097 mol). The resulting colorless solution wasstirred for 40 min until outgassing stopped. The Intermediate 4, (1.50g, 0.0092 mol) was added to the solution. After 1.25 hr the reaction wascomplete by HPLC. The reaction was diluted with CH₂Cl₂ (40 mL) andwashed with 1M HCl (40 mL), H₂O (40 mL), and saturated aqueous NaHCO₃(40 mL). The organic layer was filtered through phase separation paperand concentrated to 2.67 g of an orange oil (95% by HPLC). This oil waspurified by MPLC 0-70% EtOAc in Hexanes. Fractions with 32-42% EtOAc inHexanes contained product and were concentrated to give 2.25 g ofIntermediate 5, as a viscous yellow oil (76%).

¹H NMR (300 MHz, CDCl₃): δ 10.0 (s, 1H), 7.82 (d, 2H), 7.42 (d, 2H),3.40 (m, 2H), 3.20-3.0 (m, 3H), 2.90-2.80 (d, 1H), 2.21 (s, 3H), 1.80(m, 2H), 1.05 (s, 3H), 0.68 (s, 3H) ppm.

Example 6 Compound 13-({4-[3-oxo-3-(3,4,4-trimethyl-3b,4,4a,5-tetrahydro-1H-cyclopropa[3,4]cyclopenta[1,2-c]pyrazol-1-yl)propyl]benzyl}amino)propanoicacid

To a solution of Intermediate 5, (70 mg, 0.22 mmol) in methanol (10 mL)was added 3-aminopropanoic acid ([CAS 107-95-9] 21 mg, 0.24 mmol). Afterthe reaction mixture was stirred at RT for 2.5 h, sodiumcyanoborohydride (14 mg, 0.22 mmol) was added. After the mixture wasstirred at RT for 1.5 h, the mixture was concentrated and purified byMPLC (100% methanol in ethyl acetate) to give 30 mg of Compound I as acolorless solid.

¹H NMR (600 MHz, CD₃OD) δ 7.21-7.49 (m, 4H), 4.13 (s, 2H), 3.28-3.33 (m,3H), 3.09-3.18 (m, 2H), 2.99-3.09 (m, 2H), 2.90-2.98 (m, 1H), 2.42-2.53(m, 2H), 2.17 (s, 3H), 1.79-1.85 (m, 2H), 1.10 (s, 3H), 0.65 (s, 3H).

Biological Data

Compounds were synthesized and tested for S1P1 activity using the GTPγ³⁵S binding assay. These compounds may be assessed for their ability toactivate or block activation of the human S1P1 receptor in cells stablyexpressing the S1P1 receptor.

GTP γ³⁵5 binding was measured in the medium containing (mM) HEPES 25, pH7.4, MgCl₂ 10, NaCl 100, dithitothreitol 0.5, digitonin 0.003%, 0.2 nMGTP γ³⁵S, and 5 μg membrane protein in a volume of 150 μl. Testcompounds were included in the concentration range from 0.08 to 5,000 nMunless indicated otherwise. Membranes were incubated with 100 μM5′-adenylylimmidodiphosphate for 30 min, and subsequently with 10 μM GDPfor 10 min on ice. Drug solutions and membrane were mixed, and thenreactions were initiated by adding GTP γ³⁵S and continued for 30 min at25° C. Reaction mixtures were filtered over Whatman GF/B filters undervacuum, and washed three times with 3 mL of ice-cold buffer (HEPES 25,pH7.4, MgCl₂ 10 and NaCl 100). Filters were dried and mixed withscintillant, and counted for ³⁵S activity using a β-counter.Agonist-induced GTP γ³⁵S binding was obtained by subtracting that in theabsence of agonist. Binding data were analyzed using a non-linearregression method. In case of antagonist assay, the reaction mixturecontained 10 nM S1P in the presence of test antagonist at concentrationsranging from 0.08 to 5000 nM.

Table 1 shows activity potency: S1P1 receptor from GTP γ³⁵S: nM, (EC₅₀).

TABLE 1 S1P1 IUPAC name EC₅₀ (nM)3-({4-[3-oxo-3-(3,4,4-trimethyl-3b,4,4a,5-tetrahydro-1H- 262cyclopropa[3,4]cyclopenta[1,2-c]pyrazol-1-yl)propyl]benzyl}amino)propanoic acid

What is claimed is:
 1. A compound represented by Formula I, itsenantiomers, diastereoisomers, tautomers or a pharmaceuticallyacceptable salt thereof,

wherein: R is H, optionally substituted C₁₋₆ alkyl, —OC₁₋₃ alkyl,halogen, optionally substituted C₆₋₁₀ aryl, optionally substitutedheterocycle, CN, NO₂, C(O)R⁵, NR⁶R⁷ or OH; R¹ is H, halogen, optionallysubstituted C₁₋₆ alkyl, halogen, optionally substituted C₆₋₁₀ aryl,optionally substituted heterocycle; R² is H, halogen, optionallysubstituted C₁₋₆ alkyl, halogen, optionally substituted C₆₋₁₀ aryl,optionally substituted heterocycle; R³ is H, optionally substituted C₁₋₆alkyl, NR⁶R⁷ or hydroxyl; R⁴ is H, optionally substituted C₁₋₆ alkyl; R⁵is H, OH or optionally substituted C₁₋₆ alkyl; a is 0, 1, 2 or 3; b is0, 1, 2 or 3; X is S, O, NR⁶ or CHR⁷; R⁶ is H, optionally substitutedC₁₋₆ alkyl; R⁷ is H, optionally substituted C₁₋₆ alkyl; Y is OPO₃H₂,carboxylic acid, PO₃H₂, —P(O)MeOH, —P(O)(H)OH, NR⁸R⁹ or OR¹⁰; R⁸ is H,—C(O)CH₂OH or —CH₂COOH; R⁹ is H, —C(O)CH₂OH or —CH₂COOH; and R¹⁰ is H oroptionally substituted C₁₋₃ alkyl.
 2. The compound according to claim 1,wherein: R is H, optionally substituted C₁₋₆ alkyl or halogen; R¹ is H,halogen or optionally substituted C₁₋₆ alkyl; R² is H, halogen oroptionally substituted C₁₋₆ alkyl; R³ is H, NR⁶R⁷ or hydroxyl; R⁴ is Hor optionally substituted C₁₋₆ alkyl; a is 0 or 1; b is 0 or 1; X is O,NR⁶ or CHR⁷; R⁶ is H; R⁷ is H; Y is OPO₃H₂, carboxylic acid, PO₃H₂,NR⁸R⁹ or OR¹⁰; R⁸ is H, —C(O)CH₂OH or —CH₂COOH; R⁹ is H, —C(O)CH₂OH or—CH₂COOH; and R¹⁰ is H.
 3. The compound according to claim 1, wherein: Ris H; R¹ is H or optionally substituted C₁₋₆ alkyl; R² is H oroptionally substituted C₁₋₆ alkyl; R³ is H or hydroxyl; R⁴ is H ormethyl; a is 0 or 1; b is 0 or 1; X is O or NR⁶; R⁶ is H; Y is OPO₃H₂,carboxylic acid, PO₃H₂, NR⁸R⁹ or OR¹⁰; R⁸ is H, —C(O)CH₂OH or —CH₂COOH;R⁹ is H; and R¹⁰ is H.
 4. The compound according to claim 1, wherein: Ris H; R¹ is H or methyl; R² is H or methyl; R³ is H or hydroxyl; R⁴ ismethyl; a is 0 or 1; b is 0 or 1; X is O or NR⁶; R⁶ is H; Y is OPO₃H₂,carboxylic acid, PO₃H₂, NR⁸R⁹ or OR¹⁰; R⁸ is H, —C(O)CH₂OH or —CH₂COOH;R⁹ is H; and R¹⁰ is H.
 5. The compound according to claim 1, wherein: Ris H; R¹ is H or methyl; R² is H or methyl; R³ is H or hydroxyl; R⁴ ismethyl; a is 1; b is 0; X is NR⁶; R⁶ is H; and Y is carboxylic acid. 6.The compound according to claim 1, wherein: R is H, R¹ is H or methyl;R² is H or methyl; R³ is H or hydroxyl; R⁴ is methyl; a is 1; b is 0 or1; X is NR⁶; R⁶ is H; Y is OPO₃H₂, carboxylic acid, PO₃H₂, NR⁸R⁹ orOR¹⁰; R⁸ is H, —C(O)CH₂OH or —CH₂COOH; R⁹ is H; and R¹⁰ is H.
 7. Thecompound according to claim 1, wherein: R is H; R¹ is H or methyl; R² isH or methyl; R³ is H or hydroxyl; R⁴ is methyl; a is 0; b is 0 or 1; Xis O; Y is OPO₃H₂, carboxylic acid, PO₃H₂, NR⁸R⁹ or OR¹⁰; R⁸ is H,—C(O)CH₂OH or —CH₂COOH; R⁹ is H; and R¹⁰ is H.
 8. The compound accordingto claim 1, wherein: R is H; R¹ is methyl; R² is methyl; R³ is H orhydroxyl; R⁴ is methyl; a is 0; b is 0 or 1; X is O; Y is OPO₃H₂, NR⁸R⁹or OR¹⁰; R⁸ is H, —C(O)CH₂OH or —CH₂COOH; R⁹ is H; and R¹⁰ is H.
 9. Thecompound according to claim 1, wherein: R is H; R¹ is H; R² is H; R³ isH; R⁴ is methyl; a is 1; b is 0 or 1; X is NR⁶; R⁶ is H; and Y iscarboxylic acid or PO₃H₂.
 10. The compound according to claim 1 selectedfrom:


11. A pharmaceutical composition comprising as active ingredient atherapeutically effective amount of a compound according to claim 1 anda pharmaceutically acceptable adjuvant, diluent or carrier.
 12. Thepharmaceutical composition according to claim 11, wherein the compoundis selected from:


13. A method of treating a disorder associated withsphingosine-1-phosphate receptor modulation, which comprisesadministering to a mammal in need thereof, a pharmaceutical compositioncomprising a therapeutically effective amount of at least one compoundof Formula I

wherein: R is H, optionally substituted C₁₋₆ alkyl, —OC₁₋₃ alkyl,halogen, optionally substituted C₆₋₁₀ aryl, optionally substitutedheterocycle, CN, NO₂, C(O)R⁵, NR⁶R⁷ or OH; R¹ is H, halogen, optionallysubstituted C₁₋₆ alkyl, halogen, optionally substituted C₆₋₁₀ aryl,optionally substituted heterocycle; R² is H, halogen, optionallysubstituted C₁₋₆ alkyl, halogen, optionally substituted C₆₋₁₀ aryl,optionally substituted heterocycle; R³ is H, optionally substituted C₁₋₆alkyl, NR⁶R⁷ or hydroxyl; R⁴ is H, optionally substituted C₁₋₆ alkyl; R⁵is H, OH or optionally substituted C₁₋₆ alkyl; a is 0, 1, 2 or 3; b is0, 1, 2 or 3; X is S, O, NR⁶ or CHR⁷; R⁶ is H, optionally substitutedC₁₋₆ alkyl; R⁷ is H, optionally substituted C₁₋₆ alkyl; Y is OPO₃H₂,carboxylic acid, PO₃H₂, —P(O)MeOH, —P(O)(H)OH, NR⁸R⁹ or OR¹⁰; R⁸ is H,—C(O)CH₂OH or —CH₂COOH; R⁹ is H, —C(O)CH₂OH or —CH₂COOH; and R¹⁰ is H oroptionally substituted C₁₋₃ alkyl.
 14. The method of claim 13, whereinthe pharmaceutical composition is administered to the mammal to treatocular diseases, wet and dry age-related macular degeneration, diabeticretinopathy, retinopathy of prematurity, retinal edema, geographicatrophy, glaucomatous optic neuropathy, chorioretinopathy, hypertensiveretinopathy, ocular ischemic syndrome, prevention ofinflammation-induced fibrosis in the back of the eye, various ocularinflammatory diseases including uveitis, scleritis, keratitis, andretinal vasculitis; or systemic vascular barrier related diseases,various inflammatory diseases, including acute lung injury, itsprevention, sepsis, tumor metastasis, atherosclerosis, pulmonary edemas,and ventilation-induced lung injury; or autoimmune diseases andimmunosuppression, rheumatoid arthritis, Crohn's disease, Graves'disease, inflammatory bowel disease, multiple sclerosis, Myastheniagravis, Psoriasis, ulcerative colitis, autoimmune uveitis, renalischemia perfusion injury, contact hypersensitivity, atopic dermatitis,and organ transplantation; or allergies and other inflammatory diseases,urticaria, bronchial asthma, and other airway inflammations includingpulmonary emphysema and chronic obstructive pulmonary diseases; orcardiac protection, ischemia reperfusion injury and atherosclerosis; orwound healing such as but not limited to: scar-free healing of woundsfrom cosmetic skin surgery, ocular surgery, GI surgery, general surgery,oral injuries, various mechanical, heat and burn injuries, preventionand treatment of photoaging and skin ageing, and prevention ofradiation-induced injuries; or bone formation, treatment of osteoporosisand various bone fractures including hip and ankles; or anti-nociceptiveactivity, visceral pain, pain associated with diabetic neuropathy,rheumatoid arthritis, chronic knee and joint pain, tendonitis,osteoarthritis, neuropathic pains; or central nervous system neuronalactivity in Alzheimer's disease, age-related neuronal injuries; or organtransplant such as renal, corneal, cardiac or adipose tissue transplant.15. The method according to claim 13, wherein the mammal is a human.